Calpastatin in rat myoblasts: transient diminution and decreased phosphorylation depend on myogenin-directed myoblast differentiation

The formation of skeletal muscle fibers involves cessation of myoblast division, followed by myoblast differentiation and fusion to multinucleated myofibers. The myogenic regulatory factor myogenin appears at the onset of differentiation; it is required for muscle fiber formation, and cannot be replaced by other factors. The myogenin-dependent pathways and targets are not fully known. Previous studies, indicating an involvement of calpain-calpastatin and caspase in myoblast fusion, were based on the use of various inhibitors. The availability of myogenin deficient cell lines that are incapable of fusion, but regain the ability to differentiate when transfected with myogenin, provide a convenient means to study calpain-calpastatin and caspase in fusing and non-fusing myoblasts without the use of inhibitors. The differentiating wild type myoblasts exhibit decreased calpastatin phosphorylation, transient diminution in calpastatin mRNA, caspase-1 dependent diminution in calpastatin protein, and calpain-promoted proteolysis. In the myogenin-deficient myoblasts, calpastatin phosphorylation is not diminished, caspase-1 is not activated, calpastatin mRNA and protein are not diminished, and protein degradation does not occur. The myogenin-deficient myoblasts transfected with myogenin gene regain the ability to fuse, and exhibit the alterations in calpastatin and proteolysis observed in the wild type cells. Overall, the results demonstrate that the regulation of calpain in these myoblasts is independent of myogenin. In contrast, the regulation of calpastatin depends on myogenin function. The temporary diminution of calpastatin during myogenin-directed differentiation of myoblasts allows calpain activation and calpain-induced protein degradation, required for myoblast differentiation and fusion.     

Cell adhesion to collagen and decreased myogenic gene expression implicated in the control of myogenesis by transforming growth factor beta

Transforming growth factor beta 1 (TGF-beta 1) is an inhibitor of skeletal muscle myoblast differentiation. Myoblast differentiation is dependent on the expression of certain myogenic differentiation genes and is affected by cell interaction with the extracellular matrix. We have searched for events in the differentiation process of L6E9 rat myoblasts that may be involved in the inhibitory action of TGF-beta 1. Elevated expression of the myogenic differentiation gene, myogenin, which is thought to play a central role in the differentiation process, occurs 10 h after the shift of L6E9 myoblasts to differentiation medium. Elevation of myogenin mRNA is blocked by TGF-beta 1 added at the time of the shift. This effect is preceded by, and might be related to, a rapid up-regulation of junB mRNA observed in TGF-beta 1-treated L6E9 myoblasts. However, TGF-beta 1 can also block myogenic differentiation in cells transfected with the myogenin gene under the control of a constitutive SV40 viral promoter. The nature of a mechanism that could mediate the inhibitory action of TGF-beta 1 without blocking myogenin mRNA expression is suggested by the observations that (a) TGF-beta 1 upregulates type I collagen expression and deposition in L6E9 myoblasts, (b) a fibrillar type I collagen layer inhibits L6E9 myoblast differentiation, and (c) inhibition of L6E9 myoblast differentiation by a type I collagen layer occurs without a block in myogenin expression. Thus, the data suggest that inhibition of L6E9 myoblast differentiation by TGF-beta 1 may be accomplished by at least two mechanisms acting in concert. One mechanism leads to a block in the expression of myogenin whereas the other mechanism may involve TGF-beta 1-induced changes in cell adhesion that either block the action of myogenic differentiation gene products or prevent the function of other as yet unknown components of the myogenic differentiation pathway.     

高表达FoxO1抑制猪骨骼肌成肌细胞的分化

FoxO1(Forkhead box O1)是调控肌肉生长、代谢和细胞分化的重要转录因子,但其在成肌细胞分化中的作用还不甚清楚。为了研究FoxO1对哺乳动物成肌细胞分化的影响,以原代培养的长白仔猪成肌细胞作为实验材料,用2%马血清诱导分化,采用实时荧光定量PCR、Western blotting和脂质体转染等方法检测FoxO1及早期和晚期生肌调节因子MyoD和myogenin在猪成肌细胞分化过程中的表达变化。结果显示,在猪成肌细胞分化过程中,FoxO1mRNA表达量显著增加,但总蛋白量变化不显著,其磷酸化水平显著上调。同时,高表达FoxO1的猪成肌细胞中,生肌调节因子MyoD和myogenin mRNA表达受到显著抑制,而MyoD蛋白变化不显著,myogenin却显著下调(P0.05)。以上结果表明,FoxO1能够推迟猪成肌细胞的分化时间并抑制分化;同时推测,FoxO1可能通过抑制生肌调节因子的表达控制骨骼肌纤维类型的终末分化。     

The homeodomain protein Barx2 promotes myogenic differentiation and is regulated by myogenic regulatory factors

The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Here we use antisense-oligonucleotide inhibition of Barx2 expression in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, we isolated the Barx2 gene promoter and identified DNA regulatory elements that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contained binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors.     

Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/beta-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of beta-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/beta-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.     

Involvement of the ERK/MAP kinase signalling pathway in milli-calpain activation and myogenic cell migration

Recent research carried out in our laboratory has shown that IGF-1, TGF-beta1, and insulin were able to strongly stimulate myoblast migration by increasing milli-calpain expression and activity. However, the signalling pathways involved in these phenomena remain unknown. The aim of this study was to identify the signalling pathway(s) responsible for the effects of IGF-1, TGF-beta1, and insulin on myoblast migration and on milli-calpain expression and activity. For this purpose, wound healing assays were carried out in the presence of growth factors with or without specific inhibitors of ERK/MAP kinase and PI3K/Akt pathways. The results clearly showed that the inhibition of the ERK/MAP kinase pathway prevents the effects of growth factors on myoblast migration. Secondly, the expression and the activity of milli-calpain were studied in cells treated with growth factor, alone or with ERK/MAP kinase inhibitor. The results demonstrated that the up-regulation of milli-calpain expression and activity was mediated by the ERK/MAP kinase pathway. Finally, the possible implication of MyoD and myogenin, myogenic regulatory factors able to regulate milli-calpain expression, was studied. Taken together our results clearly showed that the ERK/MAP kinase signalling pathway is responsible for the effects of the three growth factors on myoblast migration and on milli-calpain expression and activity. On the opposite, the PI3K/Akt signalling pathway, MyoD and myogenin seem to be not implicated in these phenomena.     

Glucocorticoid inhibition of C2C12 proliferation rate and differentiation capacity in relation to mRNA levels of the MRF gene family

The muscle regulatory factors (MRF) gene family regulate muscle fibre development. Several hormones and drugs also affect muscle development. Glucocorticoids are the only drugs reported to have a beneficial effect on muscle degenerative disorders. We investigated the glucocorticoid-related effects on C2C12 myoblast proliferation rate, morphological differentiation, and subsequent mRNA expression patterns of the MRF genes. C2C12 cells were incubated with the glucocorticoids dexamethasone or alpha-methyl-prednisolone. Both glucocorticoids showed comparable effects. Glucocorticoid treatment of C2C12 cells during the proliferative phase reduced the proliferation rate of the cells dose dependently, especially during the third and fourth day of culture, increased MyoD1, myf-5, and MRF4 mRNA levels, and reduced myogenin mRNA level, compared to untreated control cells. Thus, the mRNA level of proliferation-specific MyoD1 and myf-5 expression does not seem to associate with C2C12 myoblast proliferation rate. Glucocorticoid treatment of C2C12 cells during differentiation reduced the differentiation capacity dose dependently, which is accompanied by a dose dependent reduction of myogenin mRNA level, and increased MyoD1, myf-5, and MRF4 mRNA levels compared to untreated control cells. Therefore, we conclude that glucocorticoid treatment reduces differentiation of C2C12 myoblasts probably through reduction of differentiation-specific myogenin mRNA level, while inducing higher mRNA levels of proliferation-associated MRF genes.     

外源性重组人睫状神经营养因子抑制成人成肌细胞的体外分化

为探讨外源性重组人睫状神经营养因子(rhCNTF)在成肌细胞分化中的作用,实验观察了0-10 ng/mlrhCNTF对成人成肌细胞体外分化的影响。结果表明,与对照组相比,2.5-10 ng/ml rhCNTF能显著抑制成肌细胞的体外分化(P<0.01),并呈量-效依赖关系,且这种抑制作用是可逆的。Western Blot分析提示,这种抑制作用伴有成肌细胞分化期特异标志myogenin和p21表达量的显著降低(P<0.01),以及成肌细胞增殖期特异标志myf5和desmin表达量的显著增加(P<0.01)。因此可以认为,外源性rhCNTF能可逆地抑制成人成肌细胞的体外分化并保持增殖。